Method and compositions providing enhanced chemiluminescence from 1,2-dioxetanes

ABSTRACT

A method and compositions including a 1,2-dioxetane and a fluorescent compound is described. In particular, enzymatic triggering of a triggerable 1,2-dioxetane admixed with a surfactant and the fluorescent compound attached to a hydrocarbon to provide a co-surfactant in a micelle or other structure providing close association of these molecules is described. The method and compositions are useful in immunoassays and in DNA probes used for various purposes.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. Pat. application Ser.No. 887,139, filed Jul. 17, 1986.

BACKGROUND OF THE INVENTION

(1) State of the Invention

The present invention relates to compositions containing a fluorescentcompound and a stable 1,2-dioxetane which can be triggered by chemicalreagents including enzymes and generate enhanced chemiluminescence. Inparticular the present invention relates to a method for significantlyenhancing the chemiluminescence which involves intermolecular energytransfer to a fluorescent compound in an organized assembly, such as amicelle, which maintains a close spacing between the dioxetane and thefluorescent compound.

(2) Prior Art

1. Mechanisms of Luminescence. Exothermic chemical reactions releaseenergy during the course of the reaction. In virtually all cases, thisenergy is in the form of vibrational excitation or heat. However, a fewchemical processes generate light or chemiluminescence instead of heat.The mechanism for light production involves thermal or catalyzeddecomposition of a high energy material (frequently an organic peroxidesuch as a 1,2-dioxetane) to produce the reaction product in a triplet orsinglet electronic excited states. Fluorescence of the singlet speciesresults in what has been termed direct chemiluminescence. Thechemiluminescence quantum yield is the product of the quantum yields forsinglet chemiexcitation and fluorescence. These quantities are oftenexpressed as efficiencies where efficient (%)=Φ×100. Energy transferfrom the triplet or singlet product to a fluorescent acceptor can beutilized to give indirect chemiluminescence. The quantum yield forindirect chemiluminescence is the product of the quantum yields forsinglet or triplet chemiexcitation, energy transfer, and fluorescence ofthe energy acceptor. ##STR1##

2. Dioxetane Intermediates in Bioluminescence. In 1968 McCapra proposedthat 1,2-dioxetanes might be the key high-energy intermediates invarious bioluminescent reactions including the firefly system. (F.McCapra Chem. Commun., 155 (1968)). Although this species is apparentlyquite unstable and has not been isolated or observed spectroscopically,unambiguous evidence for its intermediacy in the reaction has beenprovided by oxygen-18 labeling experiments (O. Shimomura and F. H.Johnson, Photochem. Photobiol., 30, 89 (1979)). ##STR2## 3. FirstSynthesis of Authentic 1,2-Dioxetanes. In 1969 Kopecky and Mumfordreported the first synthesis of a dioxetane(3,3,4-trimethyl-1,2-dioxetane) by the base-catalyzed cyclization of abeta-bromohydroperoxide. (K. R. Kopecky and C. Mumford, Can. J. Chem.,47, 709 (1969)). As predicted by McCapra, this dioxetane did, in fact,produce chemiluminescence upon heating to 50° C. with decomposition toacetone and acetaldehyde However, this peroxide is relatively unstableand cannot be stored at room temperature (25° C.) without rapiddecomposition. In addition, the chemiluminescence efficiency is very low(less than 0.1%).

Bartlett and Schaap and Mazur and Foote independently developed analternative and more convenient synthetic route to 1,2-dioxetanes.Photooxygenation of properly-substituted alkenes in the presence ofmolecular oxygen and a photosensitizing dye produces dioxetanes in highyields (P D. Bartlett and A. P. Schaap, J. Amer Chem. Soc., 92, 3223(1970) and S. Mazur and C. S. Foote, J. Amer. Chem Soc., 92, 3225(1970)). The mechanism of this reaction involves the photochemicalgeneration of a metastable species known as singlet oxygen whichundergoes 2+2 cycloaddition with the alkene to yield the dioxetane.Research has shown that a variety of dioxetanes can be prepared usingthis reaction (A. P. Schaap, P. A. Burns and K. A. Zaklika, J. Amer.Chem. Soc., 99, 1270 (1977); K. A. Zaklika, P. A. Burns, and A. P.Schaap, J. Amer. Chem. Soc., 100, 318 (1978); K. A. Zaklika, A. L.Thayer, and A. P. Schaap, J. Amer. Chem. Soc., 100, 4916 (1978); K. A.Zaklika, T. Kissel, A. L. Thayer, P. A. Burns, and A. P. Schaap,Photochem Photobiol., 30, 35 (1979); and A. P. Schaap, A. L. Thayer andK. Kees , Organic Photochemical Synthesis, II, 49 (1976)). During thecourse of this research, a polymer-bound sensitizer forphotooxygenations was developed (A. P. Schaap, A. L. Thayer, E. C.Blossey, and D. C. Neckers, J. Amer. Chem. Soc., 97, 3741 (1975); and A.P. Schaap, A. L. Thayer, K. A. Zaklika, and P. C. Valenti, J. Amer.Chem. Soc., 101, 4016 (1979)). This new type of sensitizer has beenpatented and sold under the tradename SENSITOX™ (U.S. Pat. No. 4,315,998(2/16/82); Canadian Patent No. 1,044,639 (12/19/79)). Over fiftyreferences have appeared in the literature reporting the use of thisproduct. ##STR3##

4. Preparation of Stable Droxetanes Derived from Sterically HinderedAlkenes. Wynberg discovered that photooxygenation of sterically hinderadalkenes such as adamantylideneadamantane affords a very stable dioxetane(J. H. Wieringa, J. Strating, H. Wynberg, and W. Adam, TetrahedronLett., 169 (1972)). A collaborative study by Turro and Schaap showedthat this dioxetane exhibits an activation energy for decomposition of37 kcal/mol and a half-life at room temperature (25° C.) of over 20years (N. J. Turro, G. Schuster, H. C. Steinmetzer, G. R. Faler, and A.P. Schaap, J. Amer. Chem. Soc., 97, 7110 (1975)). In fact, this is themost stable dioxetane yet reported in the literature. Adam and Wynberghave recently suggested that functionalized adamantylideneadamantane1,2-dioxetanes may be useful for biomedical applications (W. Adam, C.Babatsikos, and G. Cilento, Z. Naturforsch., 39b, 679 (1984); H.Wynberg, E. W. Meijer, and J. C. Hummelen, In Bioluminescence andChemiluminescence, M. A. DeLuca and W. D. McElroy (Eds ) Academic Press,New York, p. 687, 1981; and J. C. Hummelen, T. M. Luider, and H.Wynberg, Methods in Enzymology, 133B, 531 (1986)). However, use of thisextraordinarily stable peroxide for chemiluminescent labels requiresdetection temperatures of 150° to 250° C. Clearly, these conditions areunsuitable for the evaluation of biological analytes in aqueous mediaMcCapra, Adam, and Foote have shown that incorporation of a spirofusedcyclic or polycyclic alkyl group with a dioxetane can help to stabilizedioxetanes that are relatively unstable in the absence of thissterically bulky group (F. McCapra, I. Beheshti, A. Burford, R. A. Hann,and K. A. Zaklika, J. Chem. Soc., Chem. Commun , 944 (1977); W. Adam, L.A. A. Encarnacion, and K. Zinner, Chem. Ber., 116, 839 (1983); G. G.Geller, C. S. Foote, and D. B. Pechman, Tetrahedron Lett., 673 (1983);P. Lechtken, Chem Ber., 109, 2862 (1976); and P. D. Bartett and M. S.Ho, J. Amer. Chem. Soc., 96, 627 (1974)) ##STR4##

5. Effects of Substituents on Dioxetane Chemiluminescence. The stabilityand the chemiluminescence efficiency of dioxetanes can be altered by theattachment of specific substituents to the peroxide ring (K. A. Zaklika,T. Kissel, A. L. Thayer, P. A. Burns, and A. P. Schaap, Photochem.Photobiol., 30, 35 (1979); A. P. Schaap and S. Gagnon, J. Amer. Chem.Soc., 104, 3504 (1982); A. P. Schaap, S. Gagnon, and K. A. Zaklika,Tetrahedron Lett., 2943 (1982); and R. S. Handley, A. J. Stern, and A.P. Schaap, Tetrahedron Lett., 3183 (1985)). The results with thebicyclic system shown below illustrate the profound effect of variousfunctional groups on the properties of dioxetanes. Thehydroxy-substituted dioxetane (X=OH) derived from the2,3-diaryl-1,4-dioxene exhibits a half-life for decomposition at roomtemperature (25° C.) of 57 hours and produces very low levels ofluminescence upon heating at elevated temperatures. In contrast,however, reaction of this dioxetane with a base at -30° C. affords aflash of blue light Kinetic studies have shown that the deprotonateddioxetane (X=0⁻) decomposes 5.7×10⁶ times faster than the protonatedform (X=OH) at 25° C. ##STR5## X=0⁻ (chemiluminescent) X=OH(non-chemiluminescent)

The differences in the properties of these two dioxetanes arise becauseof two competing mechanisms for decomposition (K. A. Zaklika, T. Kissel,A. L. Thayer, P. A. Burns, and A. P. Schaap, Photochem Photobiol., 30,35 (1979); A. P. Schaap and S. Gagnon, J. Amer. Chem. Soc., 104, 3504(1982); A. P. Schaap, S Gagnon and K. A. Zaklika, Tetrahedron Lett.,2943 (1982); and R. S. Handley, A. J. Stern and A. P. Schaap,Tetrahedron Lett., 3183 (1985)). Most dioxetanes cleave by a processthat involves homolysis of the O--O bond and formation of a biradical.An alternative mechanism is available to dioxetanes bearing substituentssuch as O⁻ with low oxidation potentials. The cleavage is initiated byintramolecular electron transfer from the substituent to the antibondingorbital of the peroxide bond.

6. Chemical Triggering of Dioxetanes. The first example in theliterature is described above (A. P. Schaap and S. Gagnon, J. Amer.Chem. Soc., 104, 3504 (1982)). However, the hydroxy-substituteddioxetane and any other examples of the dioxetanes derived from thediaryl-1,4-dioxenes are far too unstable to be of use in anyapplication. They have half-lives at 25° C. of only a few hours Neitherthe dioxetane nor the precursor alkene would survive the conditionsnecessary to prepare derivatives. Further, these non-stabilizeddioxetanes are destroyed by small quantities of amines (T. Wilson, Int.Rev. Sci.: Chem., Ser. Two, 9, 265 (1976)) and metal ions (T. Wilson, M.E. Landis, A. L. Baumstark, and P. D. Bartlett, J. Amer. Chem. Soc., 95,4765 (1973); P. D. Bartlett, A. L. Baumstark, and M. E. Landis, J. Amer.Chem. Soc., 96, 5557 (1974) and could not be used in the aqueous buffersrequired for enzymatic triggering.

7. Energy-Transfer Chemiluminescence Involving Dioxetanes in HomogeneousSolution. The first example of energy-transfer chemiluminescenceinvolving dioxetanes was described by Wilson and Schaap (T. Wilson andA. P. Schaap, J. Amer. Chem. Soc., 93, 4126 (1971)). Thermaldecomposition of a very unstable dioxetane (cis-diethoxydioxetane) gaveboth singlet and triplet excited ethyl formate Addition of9,10-diphenylanthracene and 9,10-dibromoanthracene resulted in enhancedchemiluminescence through singlet-singlet and triplet-singletenergy-transfer processes, respectively. These techniques havesubsequently been used by many other investigators to determine yieldsof chemiexcited products generated by the thermolysis of variousdioxetanes (For a review, see W. Adam, In Chemical and BiologicalGeneration of Excited States, W. Adam and G. Cilento, Eds. Ch. 4,Academic Press, New York, 1982). Energy transfer in homogeneoussolution, however, requires high concentrations of the energy acceptorbecause of the short lifetimes of the electronically excited species.These high concentrations lead to problems of self-quenching andreabsorption. The present invention solves the problem by using the1,2-dioxetane and a fluorescent energy acceptor which are preferablyboth incorporated in a micelle affording efficient energy transferwithout the need for high concentrations of a fluorescer in bulksolution.

8. Enhanced Chemiluminescence from a Dioxetane Using IntermolecularEnergy in Micelles. Rates of various chemical reactions can beaccelerated by micelles in aqueous solution (See, for example: E. H.Cordes and R. B. Dunlap, Acc. Chem Res., 2,329 (1969). Catalysis resultsfrom solubilization of the substrate in the micellar pseudophase andfrom electrostatic, tydrophobic, or polarity factors Aqueous micelleshave been used to increase the rate of chemically triggered dioxetanes(A. P. Schaap, Final Technical Report to the Office of Naval Research,1987, page 16). No experiments using fluorescent compounds such asco-surfactants to enhance chemiluminescence efficiency are reported.

Several reports describe enhanced chemiluminescence from chemicalreactions in micellar environments. However, none of these make use ofenergy transfer to a fluorescent co-surfactant No stabilized dioxetaneshave been studied in micelles Goto has investigated the chemicaloxidation of a luciferin in the presence of neutral, anionic, andcationic surfactants (T. Goto and H. Fukatsu, Tetrahedron Lett., 4299(1969)). The enhanced chemiluminescence was attributed to an increase inthe fluorescence efficiency of the reaction product in the micellecompared to aqueous solution. The effect of cetyltrimethylammoniumbromide micelles on the chemiluminescent reaction of acridan esters inaqueous alkaline solution has been reported (F. McCapra, Acc. Chem.Res., 9, 201 (1976). McCapra indicates, however, that micellarenvironment does not "assist the excitation reaction". Rather, themicelles are thought to enhance the luminescent yield by decreasing therate of a competing, non-luminescent hydrolytic reaction. Similarily,Nikokavouras and Gundermann have studied the effect of micelles onchemiluminescent reactions of lucigenin and luminol derivatives,respectively (C. M. Paleos, G. Vassilopoulos, and J. Nikokavouras,Bioluminescence and Chemiluminescence, Academic Press, New York, 1981,p. 729; K. D. Gundermann, Ibid., p. 17). Shinkai observed thatchemiluminescence yields from unstable, non-isolable dioxetanes could beenhanced in micelles relative to water (S. Shinkai, Y. Ishikawa, O.Manabe, and T. Kunitake, Chem Lett , 1523 (1981). These authorssuggested that the yield of excited states may be higher in thehydrophobic core of micelles than in water.

The only reference to enhancement of enzymatically generatedchemiluminescence with surfactants involves the work of Kricka andDeLuca on the firefly luciferase system (L J Kricka and M. DeLuca, Arch.Biochem. Biophys., 217, 674 (1983)). Nonionic detergents and polymersenhanced the total light yield by increasing the turnover of the enzymeCationic surfactants such as (cetyltrimethylammonium bromide, CTAB)actually resulted in complete inhibition of the catalytic activity ofthe luciferase.

A method for enhancing the chemiluminescent yield of theluminol/peroxidase reaction by addition of 6-hydroxybenzothiazolederivatives or para-substituted phenols (G. H. G. Thorpe, L. J. Kricka,S. B. Moseley, T. P. Whitehead, Clin. Chem., 31, 1335 (1985); G. H. G.Thorpe and L. J. Kricka, Methods in Enzymology, 133, 331 (1986); and L.J. Kricka, G. H. G. Thorpe, and R. A. W. Stott, Pure & Appl. Chem., 59,651 (1987)). The mechanism for the enhancement is not known but it doesnot involve intramolecular energy transfer or intermolecular transfer toa co-micellar fluorescent surfactant.

Co-micellar fluorescent probes have been used to study the dynamicproperties of micelles (Y. Kubota, M. Kodama, and M. Miura, Bull. Chem.Soc.. Jpn., 46, 100 (1973); N. E. Schore and N J. Turro, J. Amer. ChemSoc., 6, 306 (1974); and G. W. Pohl, Z. Naturforsch , 31c, 575 (1976)).However, no examples appear in the literature of using these fluorescentmaterials to enhance chemiluminescent reactions in micelles throughenergy-transfer processes.

9. Chemiluminescent Immunoassays. There are no reports of dioxetanes asenzyme substrates or their use in enzyme-linked assays prior to thefiling date of Serial No. 87,139. Wynberg has used stable dioxetanes as"thermochemiluminescent" labels for immunoassays (J. C. Hummelen, T. M.Luider, and H. Wynberg, Methods in Enzymology, 133B, 531 (1986)). Thesedioxetanes are used to label biological materials such as proteins.Assays are subsequently conducted by heating the sample at 100° to 250°C. and detecting the thermally generated chemiluminescence. Thistechnique is distinctly different from the use of triggerable dioxetanesas enzyme substrates.

Luminol derivatives, acridinium esters and lucigenin have been employedas chemiluminescent labels for antigens, antibodies, and haptens (H. R.Schroeder and F. M. Yeager, Anal. Chem., 50, 1114 (1978); H. Arakawa, M.Maeda, and A. Tsuju, Anal. Biochem , 79, 248 (1979); and H. Arakawa, M.Maeda, and A. Tsuji, Clin. Chem., 31, 430 (1985). For reviews, see L. J.Kricka and T. J. N. Carter, In Clinical and Biochemical Luminescence, L.J. Kricka and T. J. N. Carter (Eds.), Marcel Dekker, Inc., New York ,1982, Ch. 8; L. J. Kricka, Ligand-Binder Assays, Marcel Dekker, Inc.,New York, 1985, Ch. 7; F. McCapra and I. Beheshti, In Bioluminescenceand Chemiluminescence: Instruments and Applications, Vol. I, K. Van Dyke(Ed.), CRC Press, Inc., Boca Raton, Fla., 1985, Ch. 2, Note, inparticular, the section on dioxetanes, p. 13; and G. J. R. Barnard, J.B. Kim, J. L. Williams, and W. P. Collins, Ibid, Ch. 7). Assay systemsinvolving the use of enzyme-labeled antigens, antibodies, and haptenshave been termed enzyme immunoassays. The enzyme labels have beendetected by color or fluorescence development techniques. More recently,luminescent enzyme immunoassays have been based on peroxidase conjugatesassayed with luminol/hydrogen peroxide, pyrogallol/hydrogen peroxide,Pholas dactylus luciferin, or luminol under alkaline conditions (L. J.Kricka and T. J. N. Carter, In Clinical and Biochemical Luminescence, L.J. Kricka and T. J. N. Carter (Eds.), Marcel Dekker, Inc , New York,1982, Ch. 8). No enzyme-linked assays have described dioxetanes asenzymatic substrates to generate light for detection prior to myapplication Serial No. 887,139.

10. Photographic Detection of Luminescent Reactions. Instantphotographic film and x-ray film have been used to record light emissionfrom several chemiluminescent and bioluminescent reactions (L. J. Krickaand G. H. G. Thorpe, Methods in Enzymology, 133, 404 (1986) andreferences therein See also: M. M. L. Leong, C. Milstein, and R. Pannel,J. Histochem. Cytochem , 34, 1645 (1986); R. A. Bruce, G. H. G. Thorpe,J. E. C. Gibbons, P. R. Killeen, G. Ogden, L. J. Kricka, and T. P.Whitehead, Analyst, 110, 657 (1985); J. A. Matthews, A. Batki, C. Hynds,and L. J. Kricka, Anal. Biochem , 151, 205 (1985); and G. H. G. Thorpe,T. P. Whitehead, R. Penn, and L. J. Kricka, Clin. Chem., 30, 806 (1984).No examples appear in the literature on the photographic detection ofchemiluminescence derived from chemical or enzymatic triggering ofstabilized dioxetanes prior to my application Serial No. 887,139.

OBJECTS

It is therefore an object of the present invention to provide a methodand compositions for enhancing the chemiluminescence of triggerable1,2-dioxetanes Further, it is an object of the present invention toprovide a method and compositions which can be used in immunoassays andwith enzyme linked DNA probes. These and other objects will becomeincreasingly apparent by reference to the following description and thedrawings.

IN THE DRAWINGS

FIG. 1 shows Arrhenius plot for the thermal decomposition ofphosphate-substituted dioxetane 2c in xylene.

FIG. 2 shows a plot of chemiluminescence quantum yield forbase-triggered reaction of dioxetane 2b as a function of theconcentration of cetyltrimethylammonium bromide (CTAB).

FIG. 2a shows an idealized structure of a dioxetane with a fluorescentco-surfactant and CTAB surfactant.

FIG. 3 shows a plot of chemiluminescence intensity vs. time for 10⁻⁵ Mdioxetane 2c with 100 microliters of human blood serum containingalkaline phosphatase in 2-amino-2-methyl-1-propanol (221) buffer (pH10.3) at 37° C.

FIG. 4 shows a plot of log (light intensity at plateau) vs. log(microliters of serum, 1-100) for dioxetane 2c.

FIG. 5 shows a plot of log (light intensity at 50 sec) vs. log(microliters of serum, 1-100) for dioxetane 2c.

FIG. 6 shows chemiluminescence spectra: (Curve A) chemiluminescence fromenzymatic triggering of dioxetane 2c in 221 buffer in the absence ofCTAB and fluorescent co-surfactant 3; (Curve B) energy-transferchemiluminescence from enzymatic triggering of dioxetane 2c in thepresence of CTAB and 3.

FIG. 7 shows a plot of light intensity vs. time for dioxetane 2c in 3 mLof 221 buffer with CTAB/fluorescer and 2.7×10⁻¹⁵ moles of alkalinephosphatase (Experiment D). Reagent background in the absence of enzymeis equal to intensity at time zero.

FIG. 8 shows plot of log (integrated light intensity) for time period ofzero to 3 minutes vs. log (moles of alkaline phosphatase).

FIG. 9 shows a plot of light intensity vs. time for dioxetane 2c in 200microliters of 221 buffer with CTAB/fluorescer and 2.3×10⁻¹⁷ moles ofalkaline phosphatase. Reagent background in the absence of enzyme isequal to the intensity at time zero.

FIG. 10 shows a plot of log (integrated light intensity) for time periodof zero to 30 minutes vs. log (moles of alkaline phosphatase).

FIG. 11 is a photographic detection of chemiluminescence from dioxetane2c using ASA 3000 Polaroid Type 57 film. Solutions of 221 buffer (100microliters) containing alkaline phosphatase, dioxetane, Mg(OAc)₂, CTAB,and fluorescein surfactant 3 were incubated in Dynatech Immulon™ wellsfor 1 hour at 37° C. and then photographed at that temperature for 15minutes. Quantitites of alkaline phosphatase: A, 2700 attomol; B, 250attomol; C, 23 attomol; and D, reagent control with no enzyme (notvisible).

FIG. 12 is a photographic detection of chemiluminescence from dioxetane2c using ASA 3000 Polaroid Type 57 film Solutions of 221 buffer(100microliters) containing alkaline phosphatase, dioxetane, Mg(OAc)₂,CTAB, and fluorescein surfactant 3 were incubated in Dyntech Immulon™wells for 1 hour at 37° C. and then photographed at that temperature for30 minutes Quantities of alkaline phosphatase: A, 250 attomol; B, 23attomol; C, 2 attomol; and D, reagent control with no enzyme (notvisible).

FIG. 13 shows a plot of light intensity vs. time for dioxetane 2c in 100microliters of 221 buffer with CTAB/fluorescer and 1.3 ng S-antigen andantibody-alkaline phosphatase conjugate. Reagent background in theabsence of enzyme is equal to the intensity at time zero.

FIG. 14 shows a plot of log (integrated light intensity) for time periodof zero to 15 minutes vs. log (ng of S-antigen coated on the microwell).

FIG. 15 is a chemiluminescent assay for S-antigen using dioxetane 2c,Mg(OAc)₂, CTAB, and fluorescein surfactant 3 in 221 buffer (100microliters). Following the luminometer experiments the 7 Immulon™ wellswere incubated for 30 minutes at 37° C. and then photographed at thattemperature for 15 minutes with ASA 3000 Polaroid Type 57 film.Quantities of S-antigen from lower left to second from upper right: 112,56, 28, 14, 7, 3.5, and 1.3 ng with well containing only reagents inupper right (not visible).

FIG. 16 is a chemiluminscent assay for S-antigen. Four wells were coatedwith 50 ng of S-antigen, reacted with MAbA9-C6 monoclonal antibody,reacted with antimouse IgG-alkaline phosphatase conjugate, and thenassayed with dioxetane 2c, Mg(OAc)₂, CTAB, and fluorescein surfactant 3in 221 buffer (100 microliters). The wells were incubated for 1 hour at45° C. and then photographed at that temperature for 30 seconds with ASA3000 Polaroid Type 57 film. A control well in the center (not visible)contained only the dioxetane in the CTAB/fluorescein buffer.

GENERAL DESCRIPTION

The present invention relates to a method for generating light whichcomprises providing a fluorescent compound in closely spacedrelationship with a stable 1,2-dioxetane compound of the formula##STR6## wherein ArOX is an aryl group having an aryl ring substitutedwith an X-oxy group which forms an unstable oxide intermediate1,2-dioxetane compound when triggered to remove X by an activating agentso that the unstable 1,2-dioxetane compound decomposes and releaseselectronic energy to form light and two carbonyl containing compounds ofthe formula ##STR7## wherein X is a chemically labile group which isremoved by the activating agent to form the unstable oxide intermediate1,2-dioxetane and wherein A are passive organic groups which allow thelight to be produced and decomposing the stable 1,2-dioxetane with theactivating agent wherein the fluorescent compound accepts the electronicenergy generated upon decomposition of the unstable oxide intermediateand produces a more intense light than is produced by the triggering ofthe dioxetane alone.

In particular the present invention relates to a method for generatinglight which comprises providing a fluorescent compound in closely spacedrelationship with a stable 1,2-dioxetane compound of the formula##STR8## wherein R₁ and R₂ together and R₃ and R₄ together can be joinedas spirofused alkylene groups which can contain hetero atoms (N, S, O orP) and aryl rings, wherein at least one of R₁ and R₂ or R₃ and R₄ is anaryl group, having an aryl ring substituted with an X oxy- group whichforms an unstable oxide intermediate 1,2-dioxetane compound whentriggered to remove X by an activating agent selected from acids, bases,salts, enzymes, inorganic and organic catalysts and electron donors sothat the unstable 1,2-dioxetane compound decomposes and releaseselectronic energy to form light and two carbonyl containing compounds ofthe formula: ##STR9## wherein those of R₁, R₂, R₃ or R₄ which areunsubstituted by an X-oxy group are carbon or hetero atom containingorganic groups which provide stability for the stable 1,2-dioxetanecompound and wherein X is a chemically labile group which is removed bythe activating agent to form the unstable oxide intermediate; anddecomposing the stable 1,2-dioxetane with the activating agent whereinthe fluorescent compound accepts the electronic energy generated upondecomposition of the unstable oxide intermediate and produces a moreintense light than is produced by the triggering of the dioxetane alone.

The present invention also relates to compositions which generate lightupon triggering which comprises a fluorescent compound and a stable1,2-dioxetane of the formula ##STR10## wherein ArOX represents an arylgroup substituted with an X-oxy group which forms an unstable oxideintermediate 1,2-dioxetane compound when triggered to remove X by anactivating agent so that the unstable 1,2-dioxetane compound decomposesand releases electronic energy to form light and two carbonyl containingcompounds of the formula ##STR11## wherein X is a chemically labilegroup which is removed by the activating agent to form the unstableoxide intermediate 1,2-dioxetane and wherein A are passive organicgroups which allow the light to be produced wherein the stable1,2-dioxetane is decomposed with the activating agent and wherein thefluorescent compound accepts the electronic energy generated upondecomposition of the unstable oxide intermediate and produces a moreintense light than is produced by the triggering of the dioxetane alone.

The compositions use the same preferred dioxetanes as the method.

Further the present invention relates to a method for generating lightwhich comprises providing a fluorescent compound in closely spacedrelationship with a stable dioxetane compound of the formula: ##STR12##wherein R₁ is selected from alkyl, alkoxy, aryloxy, dialkyl or arylamino, trialkyl or aryl silyloxy and aryl groups including spirofusedaryl groups with R₂, wherein R₂ is an aryl group which can include R₁and is substituted with an X-oxy group which forms an unstable oxideintermediate 1,2-dioxetane compound when activated by an activatingagent to remove X selected from acids, bases, salts, enzymes, inorganicand organic catalysts and electron donors so that the unstable1,2-dioxetane compound decomposes and releases electronic energy to formlight and two carbonyl containing compounds of the formula: ##STR13##wherein X is a chemically labile group which is removed by theactivating agent to form the unstable oxide intermediate and wherein R₃and R₄ are selected from aryl, heteroalkyl and alkyl groups which can bejoined together as spirofused polycyclic alkyl and polycyclic arylgroups and decomposing the stable 1,2-dioxetane with the activatingagent wherein the fluorescent compound accepts the electronic energygenerated upon decomposition of the unstable oxide intermediate andproduces a more intense light than is produced by the triggering of thedioxetane alone.

Any fluorescent compound which has a lower energy for its singletexcited states compared to the excited state of the dioxetane productcan be used to enhance the chemiluminescence efficiency. A group such asa long hydrocarbon chain (preferably 8 to 20 carbon atoms) is preferablyattached to the fluorescer so that it acts as a co-surfactant in orderto incorporate the material into the organized assembly Examples offluorescers include: any fluorescent dye; aromatic compounds includingnaphthalenes, anthracenes, pyrenes, biphenyls; acridine; coumarins;xanthenes; phthalocyanines; stilbenes; furans; oxazoles; oxadiazoles;and benzothiazoles. Most preferably a surfactant which forms micelleswith the fluorescent compound is used so that the 1,2-dioxetane isadjacent to the fluorescent compound Possible surfactants are describedin Chapter 1, pages 1 to 18 of Catalysis in Micellar and MacromolecularSystems published by Academic Press, (1975). These include: zwitterion;cationic (ammonium, pyridinium, phosphonium, sulfonium salts); anionic(sulfate, sulfonate, carboxylate salts); neutral (polyoxyethylenederivatives, cyclodextrins, long chain esters, long chain amides); andnaturally occurring surfactants (lipids).

Specifically the present invention relates to a method and compositionswhich use a stable 1,2-dioxetane compound of the formula: ##STR14##wherein R₁ is selected from lower alkyl containing 1 to 8 carbon atoms,R₂ is selected from aryl, biaryl and fused ring polycyclic aryl groupswhich can be substituted or unsubstituted, and R₃ C--is selected frompolycyclic alkyl groups containing 6 to 30 carbon atoms, wherein OX isan oxy group substituted on an aryl ring which forms an unstable oxideintermediate 1,2-dioxetane compound when triggered to remove X by anactivating agent selected from acid, base, salt, enzyme, inorganic andorganic catalysts and electron donor sources and X is a chemicallylabile group which is removed by the activating agent to form theunstable oxide intermediate and wherein (I) decomposes in the presenceof an activating agent to produce light and carbonyl containingcompounds of the formula ##STR15##

Also the present invention relates to a method and compositions whichuses a stable, 1,2-dioxetane compound of the formula: ##STR16## whereinArOX is a spirofused aryl group containing a ring substituted X-oxygroup, wherein ArOX forms an unstable oxide intermediate 1,2-dioxetanecompound when triggered by an activating agent to remove X selected fromacids, bases, salts, enzymes, inorganic and organic catalysts andelectron donors, wherein X is a chemically labile group which is removedby the activating agent to form the unstable oxide intermediate1,2-dioxetane so that the unstable 1,2-dioxetane compound decomposes toform light and two carbonyl containing derivatives of the formula

    R.sub.3 C═O and --OArC═O

and wherein R₃ C--is selected from polycyclic alkyl groups containing 6to 30 carbon atoms In this structure R₁ and R₂ are joined together.

In reference to the structure: ##STR17##

(1) When R₁ is not combined with R₂ the group is preferably alkyl,alkoxy, dialkyl or arylamino trialkyl or aryl silyloxy. The alkyl groupspreferably contain 1 to 8 carbon atoms. R₁ can also be cyclic aliphaticor aryl groups, including fused ring aryl compounds, containing 6 to 14carbon atoms. When R₁ is combined with R₂ they provide an aryl groupcontaining 6 to 30 carbon atoms.

(2) R₂ is an aryl group substituted with an X oxy (OX) group. The arylcontaining group can be phenyl, biphenyl, fused phenyl and other arylgroups and can contain between 6 and 30 carbon atoms and can includeother substituents. X is any labile group which is removed by anactivating agent. The OX group can be for instance selected fromhydroxyl, alkyl or aryl carboxyl ester, inorganic oxy acid salt,particularly a phosphate or sulfate, alkyl or aryl silyloxy and oxygenpyranoside groups.

(3) R₃ and R₄ can be the same as R₁. In the following Examples, R₃ andR₄ are combined together to form a polycyclic alkylene group,particularly for ease of synthesis and comparison; however any organicgroup can be used. Preferably the polycyclic alkylene group contains 6to 30 carbon atoms.

The stable 1,2-dioxetane compounds have relatively long 1/2 lives atroom temperatures (20°-35° C.) even though they can be triggered by theactivating agent. All of the prior art compounds are either unstable atroom temperatures or require temperatures of 50° C or above in order tobe thermally decomposed which is impractical for most applications.

The activating agent may be chemical or enzymatic. In some cases (F⁻) 1equivalent is required and in others (enzymatic) only a very smallamount is used. The agents are described in any standard chemicaltreatise on the subject and include acids, bases, salts, enzymes andother inorganic, organic catalysts. The agent used will depend upon theconditions under which the stable 1,2-dioxetane is to be activated andhow labile the X group is on a particular 1,2-dioxetane. Electron donorscan be used to remove X which can include reducing agents as well aselectrical sources of electrons.

The 1,2-dioxetane decomposes to form carbonyl containing compounds andlight. An unstable 1,2-dioxetane intermediate is formed of the formula:##STR18##

In general an --ArOX substituted 1,2-dioxetanes are formed by additionof oxygen to the appropriate alkene. These alkenes are synthesizedthrough alkyl and/or aryl substituted carbonyl containing compounds ofthe formula: ##STR19## These materials are reacted in the presence oflithium aluminum hydride or other metal hydride in a polar organicsolvent, particularly tetrahydrofuran, with a transition metal halidesalt, particularly titanium chloride, and a tertiary amine base. Thereaction is generally conducted in refluxing tetrahydrofuran and usuallygoes to completion in about 4 to 24 hours.

Preparation of and Chemical Triggering of Stabilized 1,2-Dioxetanes. Itwas discovered that thermally stable dioxetanes can be triggered bychemical and enzymatic processes to generate chemiluminescence on demand(A. P. Schaap, patent application Serial No. 887,139 filed Jul. 17,1986, A. P. Schaap, R. S. Handley, and B. P. Giri, Tetrahedron Lett.,935 (1987); A. P. Schaap, T. S. Chen, R. S. Handley, R. DeSilva, and B.P. Giri, Tetrahedron Lett., 1155 (1987); and A. P. Schaap, M. D.Sandison, and R. S Handley, Tetrahedron Lett., 1159 (1987)). To do this,new synthetic procedures were developed to produce dioxetanes withseveral key features: (1) the stabilizing influence of spirofusedadamantyl groups has been utilized to provide dioxetanes that have"shelf lives" of years at ambient temperature; (2) a moiety has beenincorporated in the structure so that direct chemiluminescence from thecarbonyl cleavage product is obtained; and (3) new methods fortriggering the chemiluminescent decomposition of the stabilizeddioxetanes were provided.

The required alkenes have teen prepared by reaction of 2-adamantanonewith aromatic esters or ketones using titanium trichloride/LAH in THF(A. P. Schaap, patent application Serial No. 887,139, filed Jul. 17,1986). This is the first report of the intermolecrlar condensation ofketones and esters to form vinyl ethers using the McMurry procedure.Although McMurry had earlier investigated the intramolecular reaction ofketone and ester functional groups, cyclic ketones and not vinyl etherswere prepared by this method (J. E. McMurry and D. D. Miller, J. Amer.Chem. Soc., 105, 1660 (1983)). ##STR20##

Photooxygenation of these vinyl ethers affords dioxetanes that areeasily handled compounds with the desired thermal stability For example,the dioxetane shown below exhibits an activation energy of 28.4 kcal/moland a half-life at 25° C. of 3.8 years. Samples of this dioxetane ino-xylene have remained on the laboratory bench for several months withno detectable decomposition. ##STR21##

However, the chemiluminescent decomposition of this dioxetane can beconveniently triggered at room temperature by removal of thesilyl-protecting with fluoride ion to generate the unstable, aryloxideform which cleaves to yield intense blue light. The half-life of thearyloxide-substituted dioxetane is 5 seconds at 25° C. The spectrum ofthe chemiluminescence in DMSO exhibited a maximum at 470 nm which isidentical to the fluorescence of the anion of the ester cleavage product(methyl 3-hydroxylbenzoate) and the fluorescence of the spent dioxetanesolution under these conditions. No chemiluminescence derived fromadamantanone fluorescence appears to be produced Chemiluminescencequantum yields for the fluoride-triggered decomposition measuredrelative to the luminol standard was determined to be 0.25 (or achemiluminescence efficiency of 25%). Correction for the fluorescencequantum yield of the ester under these conditions (Φ_(F) =0.44) gave anefficiency for the formation the singlet excited ester of 57%, thehighest singlet chemiexcitation efficiency yet reported for a dioxetaneprepared in the laboratory. ##STR22##

Enzymatic Triggering of 1,2-Dioxetanes. Biological assays such asimmunoassays and nucleic acid probes involving enzymes utilize a widevariety of substrates which either form a color (chromogenic) or becomefluorescent (fluorogenic) upon reaction with the enzyme. U.S. Pat.Application Ser. No. 887,139 describes the first dioxetanes which canfunction as chemiluminescent enzyme substrates (A. P. Schaap, patentapplication filed Jul. 17, 1986; A. P. Schaap, R. S. Handley, and B. P.Giri, Tetrahedron Lett., 935 (1987); A. P. Schaap, T. S. Chen, R. S.Handley, R. DeSilva, and B. P. Giri, Tetrahedron Lett., 1155 (1987); andA. P. Schaap, M. D. Sandison, and R. S. Handley, Tetrahedron Lett., 1159(1987)). Use of these peroxides in biological systems requiresdioxetanes which are thermally stable at the temperature of theenzymatic reaction and dont undergo rapid spontaneous decomposition inthe aqueous buffers. The spirofused adamantyl dioxetanes described inthe previous paragraph meet these requirements. 1,2-dioxetanes wereprepared bearing functional groups which can be enzymatically modifiedto generate the aryloxide form. Decomposition of this unstableintermediate provides the luminescence. 1,2-dioxetanes were synthesizedwhich can be triggered by various enzymes including aryl esterase,acetylcholinesterase, and alkaline phosphatase. The phosphatase exampleis particularly significant because this enzyme is used extensively inenzyme-linked immunoassays and nucleic acid probes.

For example, enzymatic triggering by alkaline phosphatase was observedwith the phosphate-substituted 1,2-dioxetane derived from3-hydroxy-9H-xanthen-9-one and 2-adamantanone The dioxetane is thermallystable with an activation energy of 30.7 kcal/mol and a half-life at 25°C. of 12 years. The dioxetane is not only stable in organic solvents butalso shows very slow spontaneous decomposition in aqueous buffers.##STR23##

Triggering experiments were conducted using alkaline phosphatase frombovine intestinal mucosa [suspension of 5.3 mg of protein (1100 units/mgprotein) per mL in 3.2 M (NH₄)₂ SO₄ ] and the phosphate-protecteddioxetane at pH 10.3 in 0.75 M 2-amino-2-methyl- 1-propanol buffer. A 50μL aliquot (0.013 μmol) of a phosphate-dioxetane stock solution wasadded to 3 mL of the buffer at 37° C. to give a final dioxetaneconcentration of 4.2×10⁻⁶ M. Injection of 1 μL (final conc ofprotein=1.8 μg/mL) of alkaline phosphatase to the solution resulted inburst of chemiluminescence that decayed over a period of 3 min. Overthis period of time, the background luminescence from slow non-enzymatichydrolysis of the dioxetane in the buffer was only 0.2% of that producedby the enzymatic process The total light emission was found to belinearly dependent on the dioxetane concentration. The rate of decay ofthe emission is a function of enzyme concentration while the total lightemission is independent of the enzyme concentration because of turnoverof the enzyme. The chemiluminescence spectrum for thephosphatase-catalyzed decomposition was obtained at room temperature inthe buffer solution A comparison of this chemiluminescence spectrum withthe fluorescence spectrum of the spent reaction mixture and thefluorescence spectrum of the hydroxyxanthanone cleavage product in thebuffer indicates that the emission is initiated by the enzymaticcleavage of the phosphate group in dioxetane to yield the unstablearyloxide dioxetane which generates the singlet excited anion ofhydroxyxanthanone.

SPECIFIC DESCRIPTION Synthesis of 1,2-Dioxetane Compounds andFluorescent Surfactants ##STR24## Instrumentation

Nuclear magnetic-resonance (NMR) spectra were obtained on either aNicolet NT300™ or a General Electric QE300™ spectrometer as solutions inCDCl₃ with tetramethylsilane as internal standard unless notedotherwise. Infrared (IR) spectra were obtained on either a Nicolet™ or aBeckman Acculab 8™ spectrometer. Mass spectra were obtained on either aKratos™ or an AEI MS-90™ spectrometer. Ultraviolet and visibleabsorption spectra were obtained on a Varian Cary 219™ spectophotometer.Fluorescence spectra were recorded on a Spex Fluorolog™spectrophotofluorometer. Chemiluminescence spectra were measured usingthe Spex Fluorometer. Chemiluminescence kinetic and quantum yieldmeasurements were made with luminometers constructed in this laboratory.The instruments which use RCA A-31034A gallium-arsenide photomultipliertubes cooled to -78° C. and Ortec photon-counting electronics areinterfaced to Apple IIe™ and Macintosh™ computers. Elemental analyseswere performed by Midwest Microlabs, Indianapolis. Melting points weremeasured in a Thomas Hoover™ capillary melting apparatus and areuncorrected. Precision weights were obtained on a Cahn model 4700/™electrobalance.

Materials

o-Xylene was obtained from Burdick and Jackson Laboratories and used asreceived for kinetic and spectroscopic measurements. Dry DMF and DMSOwere obtained by vacuum distillation from calcium hyride. Deuteriumoxide, 1,4-dioxade-d₈, chloroform-d, fluorescein amine (isomer 1), andother chemical reagents were purchased from Aldrich Chemical Co. Samplesof alkaline phosphatase were purchased from Sigma Chemical Co. SiLica,alumina and the other solid supports were obtained from variouscommercial sources and used without further purification.

Syntheses of Alkenes

[(3-Hydroxyphenyl)methoxymethylene]adamantane (la). A 500-mL flask wasfitted with a reflux condenser, a 125-mL addition funnel, and nitrogenline. The apparatus was dried by means of a hot air gun and nitrogenpurging. Dry THF (40 mL) was added and the flask cooled in an ice bath.TiCl₃ (1.5 g, 10 mmol) was added rapidly followed by LAH (0.19 g, 5mmol) in portions with stirring. The cooling bath was removed and theblack mixture was allowed to warm to room temperature. Triethylamine(0.7 mL, 5 mmol) was added to the stirred suspension and refluxed for 15minutes. After this period, a solution of methyl 3-hydroxybenzoate (152mg, 1 mmol) and 2-adamantanone (300 mg, 2 mmol) in 20 mL of dry THF wasadded dropwise to the refluxing mixture over 15 minutes. Refluxing wascontinued for an additional 15 minutes after which the reaction wascooled to room temperature and diluted with 100 mL of distilled water.The aqueous solution was extracted with 3 ×50 mL portions of ethylacetate. The combined organic layer was washed with water, dried overMgSO₄, and concentrated. Chromatography over silica with 15% ethylacetate/hexane gave 240 mg (89%) of la as a white solid: mp 133°-4° C.;¹ H NMR (CDCl₃)δ1.64-1.96 (m, 12H), 2.65 (s, lH), 3.24 (s, lH), 3.32 (s,3H), 5.25 (s, lH, OH exchange with D₂ O), 6.70-7.30 (m, 4H), ¹³ C NMR(CDCl₃)δ28.45, 30.36, 32.36, 37.30, 39.18, 39.33, 57.82, 114.60, 116.16,122.19, 129.24, 137.24, 155.62; MS m/e (rel intensity) 271 (20, M+1),270 (100, M), 253 (7.3), 213 (35.1), 121 (41.7), 93 (9.4); Exact mass:calcd 270.1619, found 270.1616. ##STR25##

[(3-cetoxyphenyl)methoxymethylene]adamantane (lb). Hydroxy alkene la(0.75g, 2.8 mmol) was dissolved in 10 mL of CH₂ Cl₂ and pyridine (5.2 g,65.8 mmol) under N₂. The solution was cooled in an ice bath and asolution of acetyl chloride (2.6 g, 33 mmol) in 1 mL of CH₂ Cl₂ wasadded dropwise via syringe. After 5 minutes at 0° C., TLC on silica with20% ethyl acetate/hexane showed complete acetylation of la. Afterremoval of the solvent, the solid residue was washed with 30 mL ofether. The ether was washed with 3×25 mL of water, dried over MgSO₄, andevaporated to dryness. The product was chromatographed on silica using20% ethyl acetate/hexane affording 0 45g of 1b as an oil: ¹ H NMR(CDCl₃)δ1.79-1.96 (m, 12H), 2.27 (s, 3H), 2.66 (s, lH), 3.26 (s, lH),3.29 (s, 3H), 6.99-7.36 (m, 4H); ¹³ C NMR (CDCl₃) δ20.90, 28.13, 30.07,31.99, 36.99, 38.89, 39.01, 57.59, 120.34, 122.14, 126.55, 128.66,132.19, 136.90, 142.59, 150.42, 169.04; MS m/e (rel intensity) 312 (100,M), 270 (25), 255 (19.3), 213 (20.7), 163 (12.2), 121 (30.7), 43 (30);IR (neat) 3006, 2925 2856, 1725, 1600, 1438, 1362, 1218, 1100 cm⁻¹ ;Anal. Calcd. for C₂₀ H₂₄ O₃ : C, 76.92; H, 7.69, Found: C, 76.96; H,7.85. ##STR26##

[(3-Phosphatephenyl)methoxymethylene]adamantane, disodium salt (lc).

Hydroxy alkene la (500 mg, 1.58 mmol) was dissolved in 5 mL of drypyridine (dried over basic alumina). This solution was slowly added to acold mixture of 1 mL (10.7 mmol) of phosphoryl chloride and 5 mL of drypyridine at such a rate that the temperature of the reaction remainedbelow 5° C. After 30 minutes the reaction was terminated and thephosphoryl dichloridate product was poured onto a mixture of 20 g of iceand 1 mL of 10 N sodium hydroxide. The mixture was transferred to aseparatory funnel and washed with 5×30 mL portions of CH₂ Cl₂. Theproduct precipitated from the aqueous fraction after overnightrefrigeration. The solid material was washed with 3×10 mL portions ofCH₂ Cl₂ followed by 3×10 ML portions of cold water. The white solid wasthen dried under reduced pressure to give 400 mg (1.02 mmol, 64%) ofphosphorylated alkene lc: ¹ H NMR (D₂ O/p-dioxane-d₈) δ 1.67-1.83 (m,12H), 2.50 (s, lH) 3.04 (s, lH) 3.19 (s, 3H) 6.7-7.2 (m 4H) ¹³ C NMR (D₂O) δ 28.29, 30.44, 32.45, 36.99, 38.89, 57.98, 120.16, 120.85, 123.74,128.89, 133.15, 136.11, 142.68, 154.45; ³¹ P NMR (D₂ O/p-dioxane-d₈)δ1.586. ##STR27##

Synthesis of fluorescein Surfactant

5-(N-tetradecanoylaminofluorescein (3)

A solution of myristoryl chloride (2.18 g, 8.83 mmol) in THF was addedto a solution of fluoresceinamine-isomer 1 from Aldrich Chemical Co.(3.07 g, 8.85 mmol) in dry pyridine (dried over basic alumina) dropwisewith stirring at room temperature over a 12 hour period. TLC on silicawith 20% MeOH/benzene showed conversion to a less polar product. Thereaction was poured into ice water and the solid precipitate isolated byfiltration to give 4 g of solid orange material Column chromatographywith 10% MeOH/benzene afforded 500 mg of the pure product as an orangesolid: mp 185-190° C.; ¹ H NMR (CDCl₃)δ 0.88 (t, 3H), 1.27 (m, 20H),1.74 (m, 2H), 2.42 (t, 2H), 6.54-8.32 (m, 9H); ¹³ C NMR (CDCl₃) δ 13.05,22.27, 25.34, 28.90, 29.01, 29.17, 29.31, 31.61, 36.65, 101.00, 110.25,112.43, 114.93, 124.41, 126.58, 127.86, 128.77, 140.41, 147.00, 152.89,160.28, 169.96, 173.65; MS (FAB) m/e (rel intensity) 558 (10, M+1), 402(14.7), 388 (14.2), 374 (12.5), 348 (28.1), 302 (35.2), 213 (36.4), 165(36.4), 133 (50). The properties of 3 are in agreement with thosedescribed for the commercially available material from Molecular Probes,Inc. ##STR28##

Synthesis of 1-Hexadecyl-6-hydroxy-2-benzothiazamide (4)

Methyl 6-hydroxy-2-benzothiazoate (60 mg, 0.30 mmol) [see F. McCapra andZ. Razani, Chem. Commun., 153 (1976)] and 1-hexadecylamine (430 mg, 1.8mmol) were dissolved in methanol. After refluxing the solution for twodays, thin layer chromatography with 40% EtOAc/hexane showed conversionof the benzothiazoate to a less polar material. The methanol wasevaporated and the residue was then chromatographed with 2% EtOAC/hexaneto remove the excess 1-hexadecylamine. The chromatography was thencontinued with 50% EtOAC/hexane and afforded 4 as a white solid: 33 mg(27%); mp 82-4° C.; ¹ H NMR (acetone-d₆) δ 0.83 (t, 3H), 1.32 (br 26H),1.66 (m, 2H), 3.43 (t, 2H), 7.10-7.88 (m, 3H), 8.15 (br. 1H, OHexchanges with D₂ O); ¹³ C NMR (benzene-d₆) δ 14.29, 23.05, 27.08,29.58, 29.66, 29.75 29.86, 30.13, 32.28, 39.98, (acetone-d₆) δ 107.51,117.69, 125.61, 139.33, 147.80, 157 63, 160.4(, 162.12; MS (m/e (rel.intensity) 418 (M+, 33.7), 360 (14.1), 240 (61.1), 178 (77.3), 151(39.1), 97 (28.6), 83 (34.7); Exact mass: calcd. 418.2653, found418.2659 for C₂₄ H₃₈ N₂ O₂ S. ##STR29##

Preparation of 1,2-Dioxetanes

Photooxygenation procedure. Typically a 5-10 mg sample of the alkene wasdissolved in 5 mL of CH₂ Cl₂ in the photooxygenation tube. Approximately40 mg of polystyrene-bound Rose Bengal (Sensitox I) [reference to thistype of sensitizer: A. P. Schaap, A. L. Thayer, E. C. Blossey, and D. C.Neckers, J. Amer. Chem. Soc., 97, 3741 (1975)] was added and an oxygenbubbler connected. Oxygen was passed slowly through the solution for 5minutes and the apparatus immersed in a half-silvered Dewar flaskcontaining dry ice/2-propanol. The sample was irradiated with either a250 W or 1000 W sodium Lamp (General Electric Lucalox) and a UV cutofffilter while oxygen was bubbled continuously. Progress of the reactionwas monitored by TLC. A spot for the highly stable dioxetanes couldusually be detected and had a R_(f) slightly less than that of thealkene. The adamantyl-substituted dioxetanes were filtered at roomtemperature, evaporated on a rotary evaporator and recrystallized from asuitable solvent.

4-(3-Hydroxyphenyl)-4-methoxyspiro[1,2-dioxetane -3,2'-adamantane](2a).

Hydroxy alkene la (100 mg) was irradiated with the 1000W Na lamp in 8 mLof CH₂ Cl₂ at -78° C. in the presence of Sensitox I. The alkene anddioxetane on TLC using 20% ethyl acetate/hexane exhibit the same R_(f)value. Therefore, the reaction was stopped w[en a trace of the cleavageproduct began to appear. The sensitizer was removed by filtration andthe solvent evaporated. lH NMR was used to check that all of thestarting material had been oxidized Dioxetane 2a was recrystallized frompentane/benzene to give a white solid: mp 135° C.: lH NMR (CDCl₃) δ1.04-2.10 (m, 12H), 2.21 (s, lH), 3.04 (s, lH), 3.24 (s, 3H), 6.48 (s,lH, OH exchange with D20), 6.93 7.30 (m, 4H). 13C NMR (CDCl₃) δ 25.81,25.95, 31.47, 31.57, 32.27, 32.86, 33.07, 34.58, 36.30, 49.83, 95.88,112.08, 116.46, 129.34, 136.1, 156.21.

4-(3-Acetoxyphenyl)-4-methoxyspiro[1,2-dioxetane -3,2'-adamantanel(2b).Alkene lb (140 mg, 0.45 mmol) was photooxygenated in 30 mL of CH₂ Cl₂ at-78° C. with the 1000 W high pressure sodium lamp using 400 mg ofSensitox I. TLC analysis on silica gel with 20% ethyl acetate/hexaneshowed clean conversion to a more polar material in 2.5 h. NMR (CDCl₃) δ0.90-1.90 (m, 12H), 2.15 (s, lH), 2.31 (s, 3H), 3.03 (s, lH), 3.23 (s,3H), 6.61-7.45 (m, 4H) ¹³ C NMR (CDCl₃) δ 21.00, 25.82, 25.97, 31.50,31.65, 32.21, 32.80, 33.09, 34.71, 36.32, 49.92, 95.34, 111.50, 122.58,129.16, 136.42, 150.72, 169.11.

4-Methoxy-4-(3-phosphatephenyl)spiro[1,2-dioxetane-3,2'-adamantane],disodium salt (2c) Alkene lc (50mg) was photooxygenated in 2 mL of D₂O/p-dioxane-d₈ (1:1 v/v) at 10° C. with the 1000 W high pressure sodiumlamp using Sensitox I. ^(l) H NMR analysis showed clean conversion todioxetane 2c in 45 minutes. The sensitizer was removed by filtration andthe filtrate used as a stock solution for chemiluminescence experiments.¹ H NMR (D₂ O/p-dioxane-d₈ δ 0.91-1.70 (m, 12H), 2.08 (s, lH), 2.80 (s,lH) 3.07 (s, 3H), 7.00-7.26 (m, 4H); ¹³ C NMR (D₂ O/p-dioxane-d₈) δ28.95, 30.95, 32.98, 37.65, 39.53, 58.31, 120.62, 121.64, 123.55,129.31, 132.45, 136.57, 143.98, 155.30.

Chemiluminescence Kinetics Procedures

Rates for thermal decomposition of the stable dioxetanes were monitoredby the decay of chemiluminescence of aerated solutions. A cylindricalPyrex vial equipped with magnetic stir bar was filled with 3-4 mL of thereaction solvent, sealed with a Teflon-lined screw cap and placed in thethermostatted sample block of the chemiluminescence-measuringluminometer. Temperature control was provided by an external circulatingwater bath. Appropriate values for the instrument gain and optical slitsize were selected. When thermal equilibrium was reached (ca. 3minutes), an aliquot of the dioxetane stock solution sufficient toachieve a final concentration not greater than 10⁻⁴ M was added viapipette by opening the top of the luminometer or via syringe through alight-tight rubber septum located in the cover directly above the vial.The vial was sealed with a Teflon-lined screw cap to prevent evaporationwhen high temperatures were used. Measurement of the signal was begun byopening the shutter. The chemiluminescent decay was generally recordedfor at least three half-lives. Calculation of the first-order rateconstant (k) from the In (Intensity) vs. time data was performed by acomputer program utilizing a standard least-squares treatment. Thecorrelation coefficient (r) was typically at least 0.999 and k variedless than 5% between replicate samples.

Activation parameters for decomposition of the dioxetanes werecalculated from plots of In k vs. 1/T (Arrhenius eq.) or In k/t vs. 1/T(Eyring eq.) by a standard least-squares linear regression analysis. Theresults of replicate runs at 5 to 10 temperatures encompassing atemperature range of 80° to 120° C. were found to yield a straight linewith a correlation coefficient of 0.99 or better. For example, FIG. 1shows an Arrhenius plot for the thermal decomposition ofphosphate-substituted dioxetane 2 c in o-xylene with E_(a) =32.5kcal/mol and r=0.999. The half-life for 2 c at 25° C. is calculated fromthe Arrhenius equation to be 19 years.

Activation Energies for Thermal Decomposition of Dioxetanes in Xylene.

    ______________________________________                                        Dioxetane (X)                                                                           E.sub.a                                                                              Log A   k(sec.sup.-1) at 25° C.                                                           t.sub.1/2 at 25° C.                ______________________________________                                        2a (OH)   28.3   12.5    5.38 × 10.sup.-9                                                                   4.1 yrs                                   2b (OAc)  30.4   13.6    1.73 × 10.sup.-9                                                                   13 yrs                                    2c (PO.sub.3 Na.sub.2)                                                                  32.5   14.9    1.19 × 10.sup.-9                                                                   19 yrs                                    ______________________________________                                    

The above results demonstrate the extremely high stability (longhalf-life) that these types of dioxetanes exhibit before triggering withthe appropriate chemical agent or enzyme.

Determination of Chemiluminescence Quantum Yields

The chemiluminescence quantum yield (Φ_(CL)) for the decomposition ofdioxetanes is defined as the ratio of einsteins of chemiluminescenceemitted to moles of dioxetane decomposed. Sufficient energy is releasedduring the reaction from the reaction enthalpy (ΔH_(R)) plus theArrhenius activation energy (E_(a)) to populate the singlet excitedstate of one of the carbonyl cleavage products. Therefore, the maximumquantum yield is 1.0. Another parameter of interest is thechemiexcitation quantum yield (Φ_(CE)) which is defined as the ratio ofexcited states formed to dioxetane decomposed. The chemiexcitationquantum yield is related to the chemiluminescence quantum yield via thefluorescence quantum yield of the dioxetane cleavage (Φ_(F)) through theequation: Φ_(CL) =Φ_(CE) X Φ_(F).

The same procedure as those employed in the measurement of the decaykinetics was used for the determination of chemiluminescence quantumyields with the following modifications. An accurately measured aliquotof a dioxetane stock solution of known concentration was added to 3 mLof the pre-thermostatted organic solvent or aqueous buffer. The reactionwas then triggered by adding the appropriate chemical reagent or enzyme.The total light intensity was integrated by a photon-countingluminometer using an RCA A-31034A gallium-arsenide PMT cooled to -78° C.Light intensity was converted to photons by reference to a calibrationfactor based on the accurately known quantum yield of thechemiluminescent reaction of luminol with base in aerated DMSO. Theluminol reaction has been determined to have a chemiluminescence quantumyield of 0.011 (1.1%) (J. Lee and H. H. Seliger, Photochem. Photobiol.,15, 227 (1972); P. R. Michael and L. R. Faulkner, Anal. Chem., 48, 1188(1976)).

Acquisition of Chemiluminescence Spectra

Spectra of the chemiluminescence from chemically or enzymaticallytriggered dioxetanes were obtained by conducting the reaction in a 1-cmsquare quartz cuvette in the sample compartment of a Spex Fluorologspectrofluorometer at ambient temperature. Correction for the decay ofthe chemiluminescence intensity during the wavelength scan was made byaccumulating the spectrum in a ratio mode so that the observed spectrumwas divided by the signal from an auxiliary detector (EMI 9781B) whichmeasures the total signal as a function of time. The monochromatorbandpass was typically 18 nm. For weakly emitting samples, severalidentical scans were performed and added together to improve thesignal-to-noise ratio.

Chemical Triggering of Dioxetanes

1. Triggering the Chemiluminescence of Hydroxy-Substituted Dioxetane 2awith Base. Treatment of 10⁻⁴ M solution of dioxetane 2a in DMSO at roomtemperature with an excess of tetra-n-butylammonium hydroxide resultedin intense blue chemiluminescence which decayed over a period of severalminutes. The emission maximum for the chemiluminescence is 470 nm. Thefluorescence of the anion of the cleavage product (methyl3-hydroxybenzoate, MHB) is identical to the chemiluminescence spectrum.These results demonstrate that the chemiluminescence process involves:(a) base triggering to yield the unstable aryloxide form of thedioxetane, (b) subsequent cleavage of this species to generate MHB inthe singlet excited state, and (c) fluorescence of MHB to yield theluminescence with an overall quantum yield (Φ_(Cl)) of 0.25. ##STR30##

2. Catalysis of the Base-Triggered Chemiluminescence ofAcetoxy-Substituted Dioxetane 2b in Aqueous Micelles: EnhancedChemiluminescence Efficiency via Intermolecular Energy Transfer.Cationic surfactants such as cetyltrimethylammonium bromide (CTAB) canbe used to increase rates for chemical triggering of chemiluminescencefrom appropriately substituted dioxetanes in aqueous solution. Forexample, CTAB catalyzes the base-induced luminescent cleavage of theacetoxy-substituted dioxetane 2b. The dioxetane is solubilized in themicelles formed by the surfactant and NaOH is added to initiate thechemiluminescence. The electrostatic attraction of the cationic headgroup and the hydroxide anion provides the observed micellar catalysis.Typically, the experiments were carried out with [2b]=9.1 ×10⁻⁵ M,[CTAB]=2×10⁻³ M, and [OH⁻ ]=9.1×10⁻⁵ M at 37° C.

The micellar environment can lead to higher chemiluminescenceefficiencies. Although the light yields of 2a and 2b with base in DMSOare extremely high at 0.25, the yield for these dioxetanes in water isonly 8.9×10⁻⁶. The principal reason for this large decrease results fromthe fact that the cleavage product (MHB) is only weakly fluorescent inwater However, as demonstrated by the following experiment, theluminescence can be enhanced by triggering the dioxetane in a micelle.The conditions for base-triggering of 2b were the same as describedabove except that the concentration of CTAB was varied from 0 to 5×10⁻³M. FIG. 2 shows a 19-fold increase in the chemiluminescence quantumyield (Φ_(Cl) =1.7×10⁻⁴) above the critical micelle concentration forCTAB (cmc≅1×10⁻³ M). Enhanced chemiluminescence efficiency in themicellar environment is the result of increases in Φ_(F) and/or Φ_(CE).

Incorporation in the micelle of a co-surfactant with a fluorescent headgroup provides dramatically higher chemiluminescence yields for bothchemically and enzymatically triggered dioxetanes energy transfer fromthe excited cleavage product to the fluorescent surfactant can be veryefficient because both the triggerable dioxetane and the energy acceptor(fluorescer) are held in close proximity in the micelle as illustratedin FIG. 2a.

For example, CTAB micelles containing fluorescein surfactant 3 anddioxetane 2b were prepared in aqueous solution with final concentrationsof: CTAB (1.5×10⁻³ M), 3 (9×10⁻⁵ M), and 2b (9×10⁻⁵ M). Addition of baseat 37° C. resulted in intense yellow chemiluminescence rather than thenormal blue emission with a chemiluminescence efficiency of 1.4% (Φ_(CL)=0.014), an increase of 500-fold over the luminescence of 2b in theabsence of CTAB and 3.

Similar experiments were conducted with the benzothiazamide surfactant4. The chemiluminescence efficiency was 0 3% with λ_(max) at 506 nm.

Enzymatic Triggerinq of Phosphate-Substituted Dioxet 2c.

1. Triggering the Chemiluminescence of 2c with Alkaline Phosphatase fromHuman Blood Serum. A stock solution of dioxetane 2c was prepared indioxane/water (1:1 v/v) by photooxygenation of 1c. A sample of freshblood was drawn from a healthy donor and the red blood cells removed bycentrifugation to provide serum for the experiments. Treatment of 3 mLof a 10⁻⁵ M solution of 2c in 0.75 M 2-amino-2-methyl-1-propanol (221)buffer (pH 10.3) at 37° C. with 100 μL of serum led to the typicalintensity vs. time profile shown in FIG. 3 where the serum is injectedat time zero. Under these conditions the light intensity reaches aconstant level of approximately 2.3×10⁴ counts/sec. The backgroundluminescence signal from non-enzymatic hydrolysis of 2c is less than0.05% of the enzyme-generated value. The light intensity at the plateauis directly proportional to the enzyme concentration as shown by aseries of experiments using 100 to 1 μL of serum (FIG. 4) The lightintensity at any other time point can also be conveniently used toprovide a direct measure of the enzyme concentration (FIG. 5). ##STR31##

2. Triggering the Chemiluminescence of 2c with Alkaline Phosphatase fromBovine Intestinal Mucosa. Alkaline phosphatase from bovine intestinalmucosa was obtained from Sigma Chemical Co. as a suspension of 5.1 mg ofprotein per mL of 3.2 M (NH₄)2 SO₄ solution. In a typical experiment, 50μL of a 2.56×10⁻³ M stock solution of dioxetane 2c in 221 buffer wasadded to 3 mL of 221 buffer (0.75 M, pH 9.1) containing 8.0×10⁻⁴ MMg(OAc)₂ giving a final dioxetane concentration of 4.3×10⁻⁵ M. Injectionof a 10 μL aliquot of diluted enzyme into the solution at 37° C.resulted in chemiluminescence, the quantum yield of which was 3.1×10⁻⁵.As with chemical triggering, the addition of CTAB (1.13×10⁻³ ) resultsin a modest increase in Φ_(Cl) to 2.1×10⁻⁴. The kinetics of theenzymatic triggering were not significantly altered by the presence ofthe surfactant.

3. Triggering the Chemiluminescence of 2c with Alkaline Phosphatase:Enhanced Chemiluminescence Efficiency via Intermolecular Energy Transferi1 Aqueous Micelles. The efficiency of the enzyme triggeredchemiluminescence of 2c can be dramatically enhanced by incorporation ofthe fluorescein co-surfactant 3 in the micelles. Alkaline phosphataseexperiments with dioxetane 2c were conducted at 37° C. with 3 mL of asolution containing: 2c (4.3×10⁻⁵ M), 221 buffer (0.75 M, pH 9.1),Mg(OAc)₂ (8.0×10⁻⁴ M), CTAB (1.13×10⁻³ M), and fluorescein surfactant 3(5.6×10⁻⁵ M). Addition of alkaline phosphatase (Sigma, bovine intestinalmucosa) to give a final concentration of 12 pg/mL of protein resulted inchemiluminescence over a 45 minute period Integration of the lightintensity over the entire course of light emission gave Φ_(Cl) =0.015(or 1.5% chemiluminescence efficiency, a 500-fold increase compared tothe enzymatic reaction in the absence of CTAB and 3). As in the case ofchemical triggering of 2b, the chemiluminescence spectrum is alsostifted from the normal blue emission (FIG. 6, Curve A) to the typicalfluorescein emission (FIG. 6, Curve B), demonstrating the involvement ofenergy-transfer processes. It should be emphasized that simplereabsorption cf the blue light by 3 and subsequent fluorescence cannotbe the mechanism for the spectral shift as such a process would notresult in enhanced efficiency.

To test the sensitivity of this chemiluminescence method for evaluatingconcentrations of alkaline phosphatase in solution, a series ofexperiments were carried out using enzyme stock solutions prepared bydilutions of the commercial sample obtained from Sigma. A conservativeestimate of the concentration of phosphatase in the sample was made byassuming that the 5.1 mg of protein per mL in the sample was 100% purealkaline phosphatase A molecular weight of 140,000 as also used incalculating molar amounts of enzyme. The conditions were the same asdescribed above with 2c, CTAB, and fluorescer 3 except that the finalquantities cf enzyme in the 3 mL solutions were:

A=3.6×10⁻¹² moles

B=3.3×10⁻¹³ moles

C=3.0×10⁻¹⁴ moles

D=2.7×10⁻¹⁵ moles

E=2.5×10⁻¹⁶ moles

F=2.3×10⁻¹⁷ moles

Under these conditions the background chemiluminescence from the nonenzymatic hydrolysis of 2c in the buffer/co-micelle environment isextremely slow and gives rise to a constant signal of only a fewcounts/sec (FIG. 7). A typical intensity vs. time profile for enzymatictriggering with a phosphatase concentration of 2.7×10⁻¹⁵ moles in 3 mL(Experiment D) is shown in FIG. 7. The light intensity increases withtime over a period of 30-60 minutes depending on enzyme concentration.After this period the light remains constant until the dioxetane isconsumed. The pre-steady state period can be eliminated if the samplecontaining the dioxetane and enzyme is incubated at 37°-45° C. forseveral minutes before analysis with the luminometer.

Plots of either the integrated light intensity or intensities at aspecific time point vs. the quantity of enzyme give excellentcorrelations For example, FIG. 8 shows a plot of the log (totalenzymatic luminescence from time zero to 3 minutes) vs. log (moles ofalkaline phosphatase). The reproducibility of each run was better than4% and plots such as shown in FIG. 8 gave correlation coefficients of>0.99.

Enzymatic triggering experiments such as those described above were alsocarried out in Immumlon™ microtitre wells from Dynatech, Inc. made oftransparent polystyrene. The wells were used individually and placed ina light-tight holder which could be thermostatted. The chemiluminescencewas detected at the bottom of the well using a fiber optic connected tothe photon-counting luminometer described previously. This experimentalset-up allowed much smaller reaction volumes to be used. For example, aseries of experiments using dioxetane 2c, CTAB, and 3 in 200 μL of 221buffer with amounts of alkaline phosphatase ranging from 5×10⁻¹⁵ to2×10⁻¹⁸ moles (or 2 attomoles) were carried out FIG. 9 shows theintensity vs. time profile for an experiment with 2.3×10-17 moles ofenzyme. A more realistic assumption for the purity of the enzyme samplemight be 10.% Under those conditions it is seen from FIG. 10 that thischemiluminescent technique with dioxetane 2c permits the detection ofless than 0.2 attomoles of alkaline phosphatase.

The chemiluminescence generated by the enzymatic triggering of dioxetane2c can also be detected photographically using X-ray film and instantfilm. For example FIGS. 11 and 12 show the chemiluminescence recorded onASA 3000 Polaroid™ Type 57 film. Solutions of 221 buffer (100 μL, 0.75M, pH 9.1) containing dioxetane 2c 4.3×10⁻⁵ M), Mg(OAc)₂ (8.0×10⁻⁴ M),CTAB (1.13×10⁻³ M), and fluorescein surfactant 3 (5.6×10⁻⁵ M) wereincubated in Dynatech Immulon wells in the presence of varying amountsof alkaline phosphatase using the same enzyme stock solutions listedabove. The wells were incubated for 1 hour at 37° C. and thenphotographed at that temperature for 15 minutes by placing the wellsdirectly on the film in a light-tight incubator. The light intensityrecorded on the film clearly provides a measure of the enzymeconcentration.

4. Chemiluminescent Enzyme-Linked Assays. Enzymatic triggering ofappropriately substituted dioxetanes provides an ultrasensitivedetection method for enzyme-linked biological assays. For example,phosphate-substituted dioxetane 2c can be used with enzyme linkedimmunoassays and DNA probes that utilize alkaline phosphatase as themarker for detection. Previous detection methods make use of substrateswhich develop a color or become fluorescent upon reaction with thisenzyme. The sensitivity of the chemiluminescent technique with dioxetane2c is illustrated by an enzyme-linked immunosorbant assay (ELISA) forthe retinal protein, S-antigen. Using procedures of L. A. Donoso (L. A.Donoso, C. F. Merryman, K. E. Edelberg, R. Naids, and C. Kalsow,Investigative Ophthalmology & Visual Science 26 561 (1985)), a series of7 Immulon™ wells were coated with varying amounts of S-antigen (112, 56,28, 14, 7, 3, 1.3 ng), reacted with a monoclonal antibody (MAbA9-C6)developed in mouse, and finally reacted with anti-mouse IgG coupled toalkaline phosphatase. A chemiluminescence assay of each well was thenconducted individually by adding 100 μL of 221 buffer (0.75 M, pH 9.1)containing Mg(OAc)₂ (8.0 ×10⁻⁴ M), CTAB (1.13×10⁻³ M), and fluoresceinsurfactant 3 (5.6×10⁻⁵ M). The well was placed in the micro-luminometerand equilibrated to 37° C. for 3 minutes and 10 μL of a stock solutionof dioxetane 2c in 221 buffer was injected to give a final concentrationof 2c of 1 36×10⁻⁴ M. A typical chemiluminescence intensity vs. timeprofile is shown in FIG. 13. The reagent background luminescence is verylow and constant at 15-20 counts/sec (FIG. 13). As shown in FIG. 14, Theintegrated light intensity correlates with the amount of antigen coatedon the well. Following the experiments with the luminometer, the wellscontaining the same solution were subsequently used for a photographicdetection experiment (FIG. 15). The 7 wells were placed in a holder andincubated for 30 minutes at 37° C. and then photographed at thattemperature with ASA 3000 Polaroid™ Type 57 film for 15 min in alight-tight incubator. As shown in FIG. 15, the light intensity recordedon the film also correlates with the amount of S-antigen coated on thewell. The reproducibility of the photographic assay is illustrated inFIG. 16. Four wells coated with 50 ng of antigen were treated withbuffer and dioxetane as above, incubated for 1 hour at 45° C., and thenphotographed with the film for 30 sec at that temperature It should benoted that in both photographic assays the control well containing onlybuffer solution and dioxetane is not visible, again demonstrating theextremely low background produced by non-enzymatic hydrolysis of thedioxetane.

5. Enzymatic Triggering of Hydroxy-Substituted Dioxetane 2a with Ureasein the Presence of Fluorescent Micelles. A solution was prepared using160 mg of CTAB and 12.4 mg of fluorescein surfactant 3 in 200 mL ofdistilled water. A urea solution was made by dissolving 200 mg of ureain 100 mL of distilled water with EDTA added to give a finalconcentration of 0.4 mM. The substrate solution for the ureaseexperiments was obtained by mixing 10mL of each stock solution.

Experiments were carried out with urease (Sigma) by incubation at roomtemperature for periods of 0.5 to 2 hours. Subsequent injection of 10 μLof 3×10⁻³ M dioxetane 2a produced chemiluminescence which was monitoredby the luminometer and by instant film. The intensity of theluminescence provided a measure of the concentration of urease.

In the preferred method and compositions, the dioxetane is used in anamount between about 10⁻² and 10⁻⁶ M; the surfactant in an amountgreater than about 10-4 M and the co-surfactant fluorescein surfactantin an amount between about 10⁻³ and 10⁻⁶ M. The co-surfactant quenchesitself when used alone in solution In general the molar ratio of thedioxetane to fluorescent compound is between about 1,000 to 1 and 1 to 1whether in solution or in a solid composition.

Buffers are used to adjust the pH so as to optimize enzyme activity.With the phosphate (as the X-oxy group) substituted dioxetanes the pH isbetween 9 and 10 so that non-enzymatic hydrolysis of the phosphate groupis minimized resulting in low background luminescence. 2-Methyl2-amino-1-propanal (221) is a preferred buffer. Other buffers aretris(hydroxymethyl)aminomethane and carbonate. Inorganic salts such asmagnesium acetate are also used to activate the enzyme. The buffersystem is chosen to provide maximal catalytic activity for the enzymeand an acceptor for the X-oxy group cleaved from the dioxetane, such asthe phosphate.

The present invention incorporates a stable dioxetane and fluorescentenergy acceptor, preferably in organized molecular assemblies such asmicelles affording efficient energy transfer These procedures areapplicable with other types of organized assemblies including reversedmicelles, liposomes, microemulsions, films, monolayers and polymers.

It will be appreciated that the dioxetane and acceptor can be insolution in a solvent or in a solid form such as on a film. The solidphase provides ease of positioning these molecules together.

It is intended that the foregoing description be only illustrative andthat the present invention be limited only by the hereinafter appendedclaims.

I claim:
 1. A composition which generates light upon triggering whichcomprises in admixture:(a) a fluorescent compound; and (b) a stable1,2-dioxetane of the formula: ##STR32## wherein ArOX represents an arylgroup substituted with an X-oxy group which forms an unstable oxideintermediate 1,2-dioxetane compound when triggered to remove X by anactivating agent so that the unstable 1,2-dioxetane compound decomposesand releases electronic energy to form light and two carbonyl containingcompounds of the formula: ##STR33## wherein X is a chemically labilegroup which is removed by the activating agent to form the unstableoxide intermediate 1,2-dioxetane and wherein A are passive organicgroups which allow the light to be produced wherein the stable1,2-dioxetane is decomposed with the activating agent wherein thedioxetane and the fluorescent compound are provided in a closely spacedrelationship in the presence of a surfactant, micelle, liposome,reversed micelle, microemulsion, film, including a monolayer, or polymerand wherein the fluorescent compound accepts the electronic energygenerated upon decomposition of the unstable oxide intermediate andproduces a more intense light than is produced by the triggering of thedioxetane alone.
 2. A composition which generates light upon triggeringwhich comprises in admixture:(a) a fluorescent compound; and (b) astable 1,2-dioxetane compound of the formula: ##STR34## wherein one ofR₁ and R₂ together and R₃ and R₄ together can be joined as spirofusedalkylene which can contain hetero atoms selected from the groupconsisting of N, S, O and P in addition to carbon and aryl rings,wherein at least one of R₁ and R₂ or R₃ and R₄ is an aryl groupsubstituted with an X-oxy group which forms an unstable oxideintermediate 1,2 dioxetane compound when triggered to remove X by anactivating agent selected from acids, bases, salts, enzymes, inorganicand organic catalysts and electron donors so that the unstable1,2-dioxetane compound decomposes and releases electronic energy to formlight and two carbonyl containing compounds of the formula: ##STR35##wherein those of R₁, R₂, R₃ or R₄ which are unsubstituted by an X-oxygroup are organic groups which provide stability for the stable1,2-dioxetane compound and wherein X is a chemically labile group whichis removed by the activating agent to form the unstable oxideintermediate wherein the stable 1,2-dioxetane is decomposed with theactivating agent, wherein the dioxetane and the fluorescent compound areprovided n a closely spaced relationship in the presence of asurfactant, micelle, liposome, reversed micelle, microemulsion, film,including a monolayer, or polymer and wherein the fluorescent compoundaccepts the electronic energy generated upon decomposition of theunstable oxide intermediate and produces a more intense light than isproduced by the triggering of the dioxetane alone.
 3. A compositionwhich generates light upon triggering which comprises in admixture:(a) afluorescent compound; and (b) a stable dioxetane compound of theformula: ##STR36## wherein R₁ is selected from alkyl, alkoxy, aryloxydialkylamino, trialkyl or aryl silyloxy and aryl groups includingspirofused aryl groups with R₂, wherein R₂ si an aryl group which caninclude R₁ and is substituted with an X-oxy group which forms anunstable oxide intermediate 1,2-dioxetane compound when triggered toremove X by an activating agent selected from acids, bases, salts,enzymes, inorganic and organic catalysts and electron donors so that theunstable 1,2-dioxetane compound decomposes and releases electronicenergy to form light and two carbonyl containing compounds of theformula: ##STR37## wherein X is a chemically labile group which isremoved by the activating agent to form the unstable oxide intermediateand wherein R₃ and R₄ are selected from aryl, heteroalkyl and alkylgroups which can be joined together as spirofused polycyclic alkyl andpolycyclic aryl groups wherein the stable 1,2-dioxetane is decomposedwith the activating agent, wherein the dioxetane and the fluorescentcompound are provided in a closely spaced relationship in the presenceof a surfactant, micelle, liposome, reversed micelle, microemulsion,film, including a monolayer, or polymer and wherein the fluorescentcompound accepts the electronic energy generated upon decomposition ofthe unstable oxide intermediate and produces a more intense light thanis produced by the triggering of the dioxetane alone.
 4. The compositionof claim 1 wherein the mole ratio of dioxetane to fluorescent compoundis between about 1,000 to 1 and 1 to
 1. 5. The composition of claim 1wherein the fluorescent compound is a fluorescein compound.
 6. Thecomposition of claim 1 wherein in addition a surfactant is provided inthe composition.
 7. The composition of claim 2 wherein R₁ is a methoxygroup and R₂ is a phenyl group substituted with an X-oxy group andwherein R₃ and R₄ are joined together as an adamantyl group.
 8. Thecomposition of claim 7 wherein the X-oxy group is selected fromhydroxyl, trialkyl or aryl silyloxy, inorganic oxyacid salt,oxygen-pyranoside, arylcarboxyl esters and alkylcarboxyl esters andwherein X is removed by the activating agent.
 9. The composition ofclaim 8 wherein the fluorescent compound is a fluorescein compound. 10.The composition of claim 1 wherein a cationic surfactant is provided inthe composition, wherein the fluorescent compound is chemically linkedwith a hydrocarbon chain to provide a fluorescent hydrocarbon which actsas a co-surfactant with a cationic surfactant to increase the light fromthe dioxetane and fluorescent compound and wherein the dioxetane ispresent in an amount between about 10⁻² to 10⁻⁶ M, the surfactant ispresent in an amount greater than 10⁻⁴ M and the co-surfactant ispresent in an amount between 10⁻³ to 10⁻⁶ M.
 11. The composition ofclaim 10 wherein the cationic surfactant is a cetyltrimethylammoniumsalt.
 12. The composition of claim 1 wherein the dioxetane andfluorescent compound are in a closely spaced relationship in a micelle,liposome, reversed micelle, microemulsion, film, monolayer or polymer.13. The composition of claim 1 wherein the 1,2-dioxetane has theformula: ##STR38## ps wherein X is a group reactive with the activatingagent to produce the two carbonyl containing compounds ##STR39## andlight.
 14. The composition of claim 13 wherein OX is a phosphate groupand the activating agent is alkaline phosphatase.
 15. The composition ofclaim 13 wherein in addition a surfactant is admixed in the composition.16. The composition of claim 15 wherein the surfactant is acetyltrimethylammonium salt.
 17. The composition of claim 13 wherein thefluorescent compound is a fluorescein compound.
 18. The composition ofclaim 17 wherein in addition a surfactant is admixed in the composition.19. The composition of claim 18 wherein the surfactant is acetyltrimethylammonium salt.
 20. The composition of claim 17 wherein thefluorescein compound is fluorescein chemically linked to a hydrocarbonto provide a fluorescein surfactant.
 21. The composition of claim 13wherein the fluorescent compound is selected from the group consistingof 5-N-tetradecanoylaminofluorescein and1-hexadecyl-6-hydroxy-2-benzothiazamide.
 22. The composition of claim 21wherein in addition a non-fluorescent surfactant is admixed in thecomposition.
 23. The composition of claim 22 wherein the surfactant is acetyltrimethylammonium salt.
 24. The composition of claim 1 wherein thefluorescent compound is fluorescein chemically linked to a hydrocarbonto provide a fluorescein surfactant which increases the light from thefluorescein compound and the dioxetane.
 25. The composition of claim 1wherein the fluorescent compound is selected from the group consistingof 5-N-tetradecanoylaminofluorescein and1-hexadecyl-6-hydroxy-2-benzothiazamide.
 26. The composition of claim 25wherein in addition a surfactant is admixed in the composition.
 27. Thecomposition of claim 26 wherein the surfactant is acetyltrimethylammonium salt.
 28. The composition of claim 3 wherein thefluorescent compound is fluorescein chemically linked to a hydrocarbonto provide a fluorescein surfactant.
 29. The composition of claim 3wherein the fluorescent compound is selected from the group consistingof 5-N-tetradecanoylaminofluorescein and1-hexadecyl-6-hydroxy-2-benzothiazamide.
 30. The composition of claim 29wherein in addition a surfactant is admixed in the composition.
 31. Thecomposition of claim 30 wherein the surfactant is acetyltrimethylammonium salt.
 32. The composition of claim 1 wherein thedioxetane and the fluorescent compound are provided in a closely spacedrelationship in a solid or liquid medium.
 33. The composition of claim 2wherein the dioxetane and the fluorescent compound are provided in aclosely spaced relationship in a solid or liquid medium.
 34. Thecomposition of claim 3 wherein the dioxetane and the fluorescentcompound are provided in a closely spaced relationship in a solid orliquid medium.
 35. The composition of claim 3 wherein in addition asurfactant is provided in the composition.